Isolation of mycoribnin-t6p



p 3, 1963 n. s. GOLDMAN ETAL 3,102,843

ISOLATION OF MYCORIBNIN-TGP Filed Nov. 1, 1962 PERCENT TRANSHITTANOE n & I a o o 9 Fl (3. l

5.0 5.0 7.0 WAVE LENGTH iN MIGRONS 8.0 9.0 I0.0 ll.0 |2.0 I10 4.0

WAVE LENGTH m mcaons (INFRARED ABSORPTION SPEGTRUM OF MYOORIBNlN-TGP) ABSORBANCY Fig. 2

WAVE LENGTH (Milimicra) INVENTORS (ULTRAVIOLET ABSORPTION SPECTRUM OF MYGORIBMN-TSP) DEXTER 3 GOLDMAN &

FRANK A. LORNITZO Wd-M ATTORNEY United States Patent 3,102,843 ISOLATION OF MYCORIBNIN-T6P Dexter S. Goldman, Madison, and Frank A. Lornitzo, Middleton, Wis, assiguors to the United States of America as represented by the Secretary of the Army Filed Nov. 1, 1962, Ser. No. 234,896 8 Claims. (Cl. 167-65) (Granted under Title 35, U.S. Code (1952), see. 266) The invention described herein may be manufactured and used by or for the Government for governmental purposes Without the payment to us of any royalties thereon.

This invention relates to a composition of matter which has been termed Mycoribnin-TfiP and which occurs in the H37Ra strain of lliycobaclerium tuberculosis. Mycoribnin-T6P has as one of its most important characteristics the fact that it inhibits in a noncompetitive fashion the biosynthesis of trehalose o-phosphate.

One of the objects of this invention is to isolate Mycoribnin-T6P from the H37Ra strain of Mycobacterlum tuberculosis.

Another object of this invention is to identify Mycoribruin-T6? by an ultraviolet absorption graph, molecular weight, and chemical and physical properties.

Still another object of this invention is to show the connection between the presence of Mycoribnin-T6P and the inhibition of the biosynthesis of trehalose 6-phosphate.

FIG. 1 is an infrared absorption graph of Myeoribnin- T61 FIG. 2 is an ultraviolet absorption graph of Mycon'bnin- T6P.

The H37Ra strain of Mycobacterium tuberculosis is avirulent and it is believed that the reason for this aviru- Ience is the presence of Mycoribnin-ToP. Mycoribnin- T6? would be very useful in the study of pathogenicity of various bacilli. The extreme sensitivity of the trehalose 6-phosphate forming system of virulent mycobacteria to Mycoribnin-ToP indicates that Mycoribnin-T6P may have value in the treatment of tuberculosis. It is suggested that if the cells of the virulent mycobacteria are prevented from forming the toxic glycolipid, trehalose 6-phosphate, they cannot proliferate in the host animal and will be destroyed by ordinary bodily defense mechanisms. At present, this compound has only been isolated from the H37Ra strain of Mycobactcrlum tuberculosis, but it may be present in a lesser amount in other bacilli.

Cells of the virulent mycobacteria grow in orientated cords while those of the avirulent mycobacteria grow in a random fashion. The cord factor which causes this corded growth has been shown to be trehalose 6,6 dimycolate where trehalose 6,6 dimycolate was isolated and identified as the cord factor from the virulent BCG and the human Brevannes strains of Mycobacterium tuberculosis, by H. Noll, H. Bloch, J. Asselineau and E. Lederer, Biochimlca et Biophysica Acza, volume 20, page 299 (1956). In the avirulent strains such as H37Ra Mycobacterium tuberculosis there is little or no cord factor, trehalose 6,6 dimycolate, which is the reason the avirulent strains of rnycobacteria grow in a random fashion; I. Asselineau, J. Bloch, and E. Lederer, American Review of Tuberculosis and Pulmonary Diseases, volume 76, page 853 (I953). The amount of this toxic lipid, trehalosc 6,6 dimycolate, that can be extracted from a strain of mycobacteria is roughly proportional to its viru lence.

Since the exact mechanisms by which antibiotics interfer with the growth of Mycobacterz'nm tuberculosis is by no means understood it can only be stated what the probable mechanism of action is of the substance being dealt with, Mycoribnin-TP. It is suggested that the H37Ra strain of Mycobocterium tuberculosis is avirulent because 2 it lacks trehaiose 6,6 dimycolate in its exterior coat, and further that the absence of trehalose 6,6 dirnycolate in this strain is due to the presence in this strain of the substance that hns been termed MycoribninT6P. Mycoribnin-T SP interferes with the synthesis of trehalose 6-phosphate and an essential building block in the formation of the toxic lipid, trehalose 6,6 dimycolate, is therefore not available. While the bacterial cells can grow and reproduce they do not produce the toxid material Whose presence is manifested by the diseased state, or pathogenicity.

Inhibition of the Synthesis of Trcltnlose 6-Pizosplrate by Illycorlbnin-TdP The test system employed to demonstrate the inhibition by Mycoribnin-T6P of the synthesis of trehalose 6-phosphate is made up as follows. Cell-free extracts of Mycobacterlum tuberculosis contain an enzyme which catalyzes the synthesis of trehalose 6-phosphate from glucose 6- phosphate and uridine diphospho-glucose. This reaction can be shown by mixing together a small amount of cellfree extract of Mycobacterium tuberculosis, bovine serum albumin (to protect the mycobacterial extract from inactivation), magnesium sulfate, ethylcnediaminetetraacetate, phosphate buffer of pH 6.8, glucose 6-phosphate and uridine diphospho-glucose. If such a mixture is held at 38 C. for 30 minutes and then the protein is removed by boiling the reaction mixture, trehalose -phosphate can be demonstrated in the clear liquid remaining after removal of the denatured protein. If either the mycobacterial extract, glucose 6-phosphate or uridine diphospho-glucose is omitted from the reaction mixture, trehalose 6-phosphate is not formed. Trchalose -phosphate has been identified by paper chromatography, by electrophoresis, and by chemical and enzymic degradation. In all tests for the presence of trehalose 6-phosphate, known trehalose 6- phosphate was always present as a control. That trehalose 6-phosphate is the product of this reaction is well established.

It small amounts of Mycoribnin-T6P are added to the complete reaction mixture which normally synthesizes trehalose 6-phosphate, as described above, the synthesis of trehalose 6phosphate is inhibited. The inhibition of this synthesis of trehalose o-phosphate is dependent upon the amount of Mycon'bnin-TP added. If sufiicient Mycoribnin-T6P is added the inhibition of the synthesis of he halose 6-phosphate is complete. This reaction and its inhibition in several different chemical procedures have been followed and in each case have shown that the normal product, trehalose 6-phosphate, is actually not formed in the presence of Mycoribnin-T6P. As Mycoribnin-T6P is purified the ability to inhibit the synthesis of trehalose 6-phospl1ate increases for a given weight of Mycoribnin- TSP.

Two different methods have been employed to isolate Mycoribnin-TfiP. Process II is the most satisfactory and uses the phenol method of recovery with an Eeteola cellulose column. Process I recovers Mycoribnin-TtSP by dialysis against an aqueous solution of potassium chloride.

Isolation of Mycoribnin-T 6P by Process I Cell-free extracts of the H3 7Ra strain of Mycobacterium tuberculosis are prepared by grinding 4-week-old cells. The cell-free extract is treated with aqueous phenol; soluble ribonucleic acid and Mycoribnin-TGP appear in the aqueous layer. The soluble ribonucleic acid and Myco ribnin-ToP are precipitated from the aqueous layer with alcohol and are finally obtained as an alcohol-washed dried powder. This powder is dissolved in a butter and dialyzed against an aqueous solution of potassium chloride; the soluble ribonucleic acid cannot traverse the semi-permeable membrane and is discarded. Mycoribnin-TGP is recovered from the salt solution by addition of barium acetate (0.1 volume of a saturated solution), chloroform (1 volume) and ethyl alcohol (0.1 volume). The barium salt of Mycoribnin-T6P is at the chloroform-water interface and can be isolated. The barium is removed by mixing a suspension of the barium salt with the lithium form of a strongly acid ion exchange resin. The lithium salt of Mycoribnin-T6P is removed by low temperature evaporation of the resulting solution.

Isolation of Mycoribnin-T6P by Process 11 Although a specific method for the preparation of cellfree extract on a laboratory scale is herein referred to, it is to be understood that other methods of preparing cellfree extracts from the cells of the H37Ra strain of Mycobacterium tuberculosis are potentially useful. The H37Ra strain of Mycobacterium tuberculosis is a well established strain of this bacillus and is available from the American Type Culture Collection (Catalogue Number ATCC 13325 obtainable [from the Director of Trudeau Laboratory, P.O. Box 670, Saranac Lake, New York). The following methods of preparation of the cell-free extracts and of the isolation and purification of Mycoribnin-T6P are given by way of illustration only and are not meant to be limiting.

The crude cell-free extract obtained from the grinding procedure is centrifuged three times; the first centrifugation is at 2, -0 g, the second at IQOOOXg and the third at 45,000Xg. The supernatant of each of these centrifugations is used as the starting material for the next centrifugation. Centrifugation can be accomplished in any standard laboratory equipment which will reach the necessary speeds. The supernatant fluid recovered from the last centrifugation is passed through a sub-micron membrane filter to remove any last traces of cell wall material and of intact cells. The filtrate so recovered is centrifuged at 100,0'00X g. The supernatant fluid is recovered. Mycoribru'n-TGP is obtained from this supernatant fluid by the general methods for the preparation of ribonucleic acid (RNA). The phenol method used was adapted from the phenol method of Kirby, K. 8., Biochem. J. '64, 405 (195 6), and of Gierer, A. and Achram, 6., Nature, 177, 702 (1956).

The preliminary purification of Mycoribnin-T6P begins with seventy-five (75) ml. of the crude cell-free extract obtained from the last centrifugation which was allowed to stir one hour at 0 C. with seventy-five ml. of 80% aqueous phenol (this reagent was made by mixing 20 grams of water with 80 grams of phenol). The resulting slurry was centrifuged at 0 C. and the phenol layer was separated. The phenol layer was washed twice, each time with 35 ml. of water. The water was removed by centrifuging the resulting mixture each time. The water washes were combined with the original water layer; the phenol layer was discarded. The combined water layers were extracted at room temperature with 100 ml. of ether to eliminate the phenol. The ether phase was removed and discarded. Two (2) grams of potassium acetate were dissolved in the aqueous fraction. This aids in the formation of layers in the extraction procedure and in the subsequent precipitation of Mycoribnin-ToP. The aqueous solution was washed twice, each time with 100 ml. of ethyl ether. The ether layers were removed and discarded. At this point in the procedure, the aqueous solution is clear and light yellow in color. The aqueous solution (115 ml.) is cooled to approximately 0' C. before the start of the next step. Absolute ethyl alcohol (230 ml.) was then added to the aqueous solution; constant thorough stirring was employed. The addition of alcohol required 5 to minutes. The temperature was kept as close to 0 C. as possible during the addition of the alcohol. The resulting mixture was stirred for an additional 20 minutes. The slurry was then centrifuged for 20 minutes at 15, 000 g. The supernatant fluid was carefully poured off and dis carded. The precipitate was removed from the sides of the centrifuge container and mixed in 30 ml. of 75% aqueous ethyl alcohol. The resulting mixture was centrifuged for 20 minutes at 30,000 g. The residue was washed twice more in the same manner this time using aqueous ethyl alcohol. The alcohol and washes were discarded in each case. No phenolic substances (bromine test) could be detected in the last two ethyl alcohol washes. After the alcohol washes were completed the precipitate was dried in a vacuum desiccator for 48 hours at room temperature. The yield of crude Mycoribnin- T6P was 380 milligrams.

Purification of Mycoribnin T6P is accomplished by putting the dried crude RNA-Mycoribnin-TtSP preparation in suspension in 40 ml. of 0.002 M aqueous ammonium carbonate. Any insoluble material was removed by centrifugation and discarded. The clear aqueous supernatant fluid has a total absorbancy of 2,400 at 260 mg; the ratio of the absorbancy at 260 m to the absorbancy at 280 m was 1.90 to 2.00. A 7.5 x 22 centimeter column of Ecteola cellulose as described in Peterson, E. A. and Sober, H. A., I. Am. Chem. Soc., 78, 751 (1956), was prepared; the absorbent was washed until it was free from chloride. Thirty-six (36) m. of the 40 ml. of the aqueous RNA-Mycoribnin-T6P solution obtained above was diluted with water and concentrated aqeous carbonate (of pH 8.5) to a final volume of 200 m1. and a final ammonia concentration of 6.5 M. The resulting mixture was poured onto the top of the column of cellulose and the solution was allowed to run into the column. The Mycoribnin-T6P was eluted from the column with 6.5 M ammonium carbonate (pH 8.5). The initial rate of the elution was 2 ml. per minute for the first 200 ml. of the eluting agent and then 10 ml. per minute. Ultraviolet absorbing material appears after approximately 200 ml. of eluting agent has come through the column. The elution of Mycoribnin-T6P continues 'for the next 800 to 1,000 ml. of the eluting agent. That portion of the eluting agent in which the ultraviolet absorbency rising from 0 to its maximum contains about 30% of the original Mycoribnin-T6P activity; purification of Mycoribnin-T6P from this portion of the eluate is difiicult. The second portion of the eluting, that portion in which the absorbancy at 260 mu falls from its maximum absorbency back toward 0, has a total ultraviolet absorbancy at 260 m of about 22 and contains about 40% of the original Mycoribnin-TGP activity. This fraction is retained. The eluate is evaporated in a rotary evaporator at 37 to 40 C.; evaporation is continued to dryness. The crystalline mass so obtained is dissolved in 50 ml. of water and re-evaporated to dryness. This is repeated twice more. Carbamate is destroyed and dissolved CO is removed after dissolving the final residue in water by adding a strong cation exchanger, Dowex 50 (X-S) H until the pH remains below 3. Nitrogen gas is bubbled through the resulting mixture for several minutes. The pH is brought back to 6.5 with aqueous potassium hydroxide. Sediment and resin is removed by centrifugation of the mixture at 2,000X g. A portion of the Mycoribnin-T6P activity is lost during the resin treatment. Most of this activity is recovered if a small amount of cobalt is added to the assay mixture. It has been found that cobalt at 1.0 M is suflicient to completely reactivate the Mycoribnin-T6P preparation. Other trace divalent metals do not reactivate the Mycoribnin-T6P. The loss of Mycoribnin- T6P activity during the resin treatment may possibly be due to a chemical change involving an opening and subsequent closing of the heterocyclic ring of the nucleotide bases.

Properties of Myc0ribnin-T6P The following are the chemical and physical properties of Mycoribnin-T6P which identify this compound which at present has an unknown chemical structure:

(a) In crude cell-free extracts Mycoribnin-T6P is stable to dialysis against water but is rapidly destroyed by exposure to phosphate.

(b) The enzyme responsible for the destruction of Mycoribnin-TGP in the presence of phosphate is most probably a polynucleotide phosphorylase.

(c) My-coribnin-T6P is destroyed by treatment with a strong acid or alkali.

(d) Mycoribnin-ToP is insoluble in chloroform and may be purified by ion exchange chromatography.

(e) Purified Myeoribnin-T6P in the presence of high concentrations of salts passes through a semi-permeable membrane. In the absence of salt this migration is not observed.

(1) Purified Mycoribnin-TGP is destroyed by ribonuclease but not by deoxyribonuclease.

(g) Mycoribnin-T6P shows an ultraviolet absorption spectrum typical of nucleotides and may be purified, in part by techniques commonly employed in nucleotide work.

(/2) The evidence gathered to date indicates that Mycoribnin-T6i is a low molecular weight polyribonucleotide.

(i) Mycoribnin-TGP has, so far, been found only in the avirulent H37Ra strain of Mycobacrerium tuberculosis but evidence exists that it may be present, although in much lower concentrations, in other mycobacteria.

(j) Mycoribnin-TEP inhibits, in a noncompetitive fashion, the biosynthesis of trehalose 6-phosphate.

(k) The infrared spectrum of Mycoribnin-T6P suspended in Nujol mull is shown in FIG. 1. There are no absorption bands at wavelengths below p. A strong absorption band is noted at 5.7 A pair of diffuse absorption bands is found at 6.6 and 69g. In the region of 8.8 to 9.21s there is a broad absorption band followed by a shoulder at 9.6 and 9.8,u.

(I) The ultraviolet absorption spectra of Mycoribnin- T61, in aqueous solutions, are reproduced in FIG. 2.

(1) At; pH 7.0, 12%,, is as follows:

24lm =5 .65 265rnn=13.8 272mu=13.0

(2) At pH 1.0, El,,, is as follows:

251mrt=9.59 267m;r=7.40 278rn,u=5.22

(m) The molecular weight of Mycoribnin-TGP is less than 5,000 (sedimentation analysis). The molecular weight, calculated from periodate oxidation of ribose released by enzyrnic hydrolysis, is 2.1001-400.

(n) In the neutral potassium salt from Mycoribnin- T6P contains the following approximate proportions of the various elements: C=26.2%, H=5.l%, O=50=.8%, N=l.7%, P:8.8%, K=l0.7%. The empirical formula of the neutral potassium salt of Mycoribnin-T6P is: 17 4fl 2-t 2 2)n- We claim:

1. Mycoribnin-TSP, a substance occurring in the avirulent strain of H37Ra Mycobacterium tuberculosis, said substance being effective in inhibiting in a noncompetitive fashion, the bi-osynthesis of trehalose 6-phosphate, said substance in cell-free form being stable to dialysis against water but being rapidly destroyed by exposure to phosphate, said substance being destroyed by treatment with a strong acid, said substance being destroyed by treatment with a strong alkali, said substance being insoluble in chloroform, said substance capable of being purified by ion exchange chromatography, said substance in its purified state being destroyed by ribonuclease but not by deoxyribonuclease, said substance in the presence of high concentrations of salts being capable of passing through a semi-permeable membrane, but in the absence of salt being unable to migrate through said semi-permeable membrane, said substance having a molecular weight of 2,1001400 and an elemental analysis in the neutral potassium salt form of C:26.2%, 0:50.895, H:5.l%, N:l.7%, P=8.8%, K=l0.7%, said substance having an empirical formula in its neutral potassium salt form of: (C I-I O NP KQ said substance having an infrared absorption spectrum as shown in FIG. 1 and having a strong infrared absorption band at 5.755, a pair of diffuse absorption bands at 6.6;.0 and 6.8 and a broad absorption hand between 8.8 and 9.8 said broad absorption band being followed by a shoulder at 9.6 and 9.8a, and said substance having an ultraviolet absorption spectra as shown in FIG. 2.

2. A process for producing the lithium salt of Mycoribnin-T6P comprising:

(a) grinding cells of the H37Ra strain of Mycolmcterium tuberculosis,

(b) treating the extract from said ground cells with aqueous phenol,

(c) precipitating with alcohol the ribonucleic acid and Mycoribnin-ToP from the aqueous layer of said treated extract,

(:1) washing with alcohol, drying and buffering said precipitate,

(c) dializing said dried precipitate against an aqueous solution of potassium chloride,

(f) recovering Mycoribnin-ToP from said dialized potassiurn chloride solution by the addition of barium acetate, chloroform, and ethyl alcohol,

(g) isolating the barium salt of Mycoribnin-TfiP at the chloroform-water interface,

(h) adding to said barium salt of Myooribnin-Tol the lithium form of a strongly acid ion exchange resin,

(1) evaporating to a powder the lithium salt of Mycoribnin-T6P produced by said ion exchange resin.

3. A process for producing the lithium salt of Mycoribnin-T6P comprising:

(a) grinding cells of the H3721 strain of Mycobacterium tuberculosis,

(11) treating the extract from said ground cells with aqueous phenol,

(c) precipitating with alcohol the ribonucleic acid and Mycoribnin-T6P from the aqueous layer of said treated extract,

(a?) washing said precipitate with alcohol,

(2) drying said precipitate to a powder,

(f) dissolving said powder in a buffer,

(g) dializing said dissolved powder against an aqueous solution of potassium chloride,

(h) recovering Mycoribnin-T6P from the salt solution by the addition of barium acetate (0.1 volume of saturated solution), chloroform (1 volume) and ethyl alcohol (0.1 volume),

(1') isolating the barium salt of Mycoribnin-T6P at the chloroform-water interface,

(j) removing the barium by mixing a suspension of the barium salt with the lithium form of a. strongly acid ion exchange resin,

(k) recovering the lithium salt of Mycoribnin-T6P by low temperature evaporation.

4. A process for producing Mycoribnin-T6P compris- (a) grinding cells of the H37Ra strain of Mycobacteritmz tuberculosis,

(11) centrifuging extract from said ground cells,

(c) filtering the supernatant fluid from said centrifugation with a submicron membrane filter,

(d) centrifuging the recovered filtrate,

(e) recovering Mycoribnin-ToP from the supernatant fluid of said centrifuged filtrate by ribonucleic acid recovery methods.

5. A process for producing Mycoribnin-T6P comprising:

(a) grinding cells of the H37Ra strain of Mycobacterium tuberculosis,

(b) centrifuging the extract from said grinding a plurality of times using the supernatant of each centrifugation as the starting material for the next centrifugation,

(c) passing the supernatant fiuid from the last of said oentrifugations through a submicron membrane filter, said filter removing any last traces of cell Wall material,

(d) centrifuging the recovered filtrate and retaining the supernatant fluid,

(e) recovering Mycoribnin-T6P from said supernatant fluid by ribonucleic acid recovery methods.

6. A process of producing Mycoribnin-T6P as claimed in claim 4 wherein the method of recovery consists of:

(a) washing and centrifuging a pluality of times the crude cell free extract with aqueous phenol,

(b) cooling, washing, and centrifuging the aqueous layer with ethyl alcohol to give a precipitate,

(c) placing said precipitate in suspension with aqueous ammonium carbonate and centrifuging,

(d) bringing said centrifuged solution to an ammonia concentration of 6.5 M,

(e) eluting said solution through an Ecteola cellulose column,

(1) evaporating to a crystalline mass the portion of the elute coming through said column after an absorbancy of the elute reaches a maximum of 260 m (g) redissolving said crystalline mass in water and reevaporating a plurality of times,

(It) adding a strong cation exchanger until the pH is below 3,

(i) bubbling nitrogen gas through the resulting mixture,

(j) adding aqueous potassium hydroxide until the pH (k) removing sediment and residue by centrifugation and adding a solution of cobalt to produce pure activated Mycoribnin-T6P.

7. A process for producing Mycoribnin-T6P as claimed in claim 4 wherein the method of recovery consists of:

((1) adding aqueous phenol to the crude cell-free extract obtained from the last of said centrifugation, (b) centrifuging and washing phenol layer a plurality of times,

() extracting phenol with ether, said extraction including an additive of potassium acetate to aid in the formation of layers,

(d) cooling and Washing the aqueous layer of said extraction with ethyl alcohol,

(e) stirring and centrifuging the resulting mixture,

(f) mixing the precipitate from said centrifugation with alcohol and centrifuging to reprecipitatc,

(g) drying said precipitate from said reprecipitation,

(h) placing this precipitate in suspension with aqueous ammonium carbonate,

(1') centrifuging out unwanted insoluble material,

(I) diluting said centrifuged solution with ammonium carbonate until ammonium concentration 6.5 M,

(k) eluting said solution through an Ecteola cellulose column,

(I) evaporating the portion of the elute following a maximum absorbancy of 260 my to a crystalline mass,

(m) redissolving said crystalline mass in water and re-evaporating a plurality of times,

(n) adding a strong cation exchanger until the pH is below 3,

(0) bubbling nitrogen gas through the resulting mixture,

in claim 4 wherein the method of recovery consists of:

(a) stirring one hour at 0 C. a mixture of crude cellfree extract obtained from the last centrifugation with aqueous phenol,

(b) centrifuging the slurry of said mixture at 0 C.,

(c) separating the phenol layer of said mixture,

(d) washing said phenol layer twice with water, said water being removed each time by centrifuging the resulting mixture,

(e) combining the washes and the original water layers,

(f) extracting the phenol in said water layers at room temperature with ether,

(g) dissolving potassium acetate in the aqueous fraction to aid in the formation of layers,

(h) washing said aqueous solution twice with ethyl ether,

(i) cooling said Washed aqueous solution to approximately 0 C.,

(j) adding absolute ethyl alcohol to said aqueous solution,

(k) stirring the alcohol mixture for 20 minutes,

(I) centrifuging the resulting slurry for 20 minutes,

(m) mixing the precipitate from said centrifugation with aqueous ethyl alcohol,

(n) centrifuging the alcohol and precipitate mixture for 20 minutes,

(0) drying the precipitate from said centrifugation to produce crude Mycoribnin-T6P,

(p) placing said dried crude Mycoribnin-TGP in suspension with ammonium carbonate,

(q) removing all unwanted insoluble material by centrifugation,

(r) diluting said Mycoribnin-T6P solution with ammonium carbonate until final ammonia concentration is 6.5 M,

(s) running the resulting mixture Ecteola cellulose column,

(t) eluting the column with 6.5 M ammonium carbonate having a pH of 8.5 and retaining the portion of the elute coming through the column when the ab sorbency falls from a maximum of 260 m to 0,

(u) evaporating the retained eluate to a crystalline mass,

(v) dissolving said crystalline mass in water and reevaporating twice more,

(w) adding a strong cation exchanger until the pH is below 3 to destroy the carbamate and dissolved CO (x) bubbling nitrogen gas through the resulting mixture,

(y) bringing the pH back to 6.5 with aqueous potassium hydroxide,

(z) removing sediment and resin by centrifugation,

and

(aa) adding 1.0 10- M cobalt solution to reactivate any opened bonds in the nucleotide bases, said addition of cobalt producing pure activated Mycoribnin- T6P.

through an No references cited. 

1. MYCORIBNIN-T6P, A SUBSTANCE OCCURRING IN THE AVIRULENT STRAIN OF H37RA MYCOBACTERIUM TUBERCULOSIS, SAID SUBSTANCE BEING EFFECTIVE IN INHIBITING IN A NONOCOMPETITIVE FASHION, THE BIOSYNTHESIS OF TREHALOSE 6-PHOSPHATE, SAID SUBSTANCE IN CELL-FREE FORM BEING STABLE TO DIALYSIS AGAINST WATER BUT BEING RAPIDLY DESTROYED BY EXPOSURE TO PHOSPHATE, SAID SUBSTANCE BEING DESTROYED BY TREATMENT WITH A STRONG ACID, SAID SUBSTANCE BEING DESTROYED BY TREATMENT WITH A STRONG ALKALI, SAID SUBSTANCE BEING INSOLUBLE IN CHLOROFORM, SAID SUBSTANCE CAPABLE OF BEING PURIFIED BY ION EXCHANGE CHROMATOGRAPHY, SAID SUBSTANCE IN ITS PURIFIED STATE BEING DESTROYED BY RIBONUCLEASE BUT NOT BY DEOXYRIBONUCLEASE, SAID SUBSTANCE IN THE PRESENCE OF HIGH CONCENTRATIONS OF SALTS BEING CAPABLE OF PASSING THROUGH A SEMI-PERMEABLE MEMBRANE, BUT IN THE ABSENCE OF SALT 